Role of nebulized colistin as a substitutive strategy against nosocomial pneumonia caused by CR-GNB in intensive care units: a retrospective cohort study

Background Adverse reactions, especially nephrotoxicity, are great concerns of intravenous colistin treatment. The role of substitutive nebulized colistin in treating nosocomial pneumonia caused by carbapenem-resistant Gram-negative bacterial (CR-GNB) in critically ill patients remains unknown. Methods This retrospective study enrolled patients with nosocomial pneumonia caused by colistin-susceptible CRGNB in the intensive care unit (ICU) without intravenous colistin treatment. Patients were categorized based on whether substitutive nebulized colistin was used alongside other intravenous antibiotics. Clinical responses and mortality rates were compared between the two groups in the original and propensity score (PS)-matched cohorts. This study aimed to investigate the clinical effectiveness of substitutive nebulized colistin in treatment outcomes of nosocomial pneumonia caused by CR-GNB. The impact of dosing strategy of nebulized colistin was also explored. Results In total, 343 and 214 patients with and without substitutive nebulized colistin, respectively, were enrolled for analysis. In the PS-matched cohort, clinical failure rates on day 7 (22.6 vs. 42.6%, p = 0.001), day 14 (27.0 vs. 42.6%, p = 0.013), and day 28 (27.8 vs. 41.7%, p = 0.027) were significantly lower in patients with nebulized colistin. In multivariate analysis, nebulized colistin was an independent factor associated with lower day 14 clinical failure (Original cohort: adjusted odds ratio (aOR) 0.45, 95% confidence interval (CI) 0.30–0.67; PS-matched cohort: aOR 0.48, 95% CI 0.27–0.87). There were no differences in clinical failure rate and mortality rate between patients receiving high (> 6 MIU/day) and low (≤ 6 MIU/day) dose nebulized colistin in the PS-matched cohort. Conclusions In ICU-admitted patients with nosocomial pneumonia caused by colistin-susceptible CRGNB, substitutive nebulized colistin was associated with better clinical outcomes. Supplementary Information The online version contains supplementary material available at 10.1186/s13613-022-01088-4.


Background
Nosocomial pneumonia, including hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP), are the leading causes of morbidity and mortality in the intensive care unit (ICU) [1,2]. Nosocomial pneumonia caused by carbapenem-resistant Gram-negative bacteria (CR-GNB) is a growing concern worldwide due to limited treatment options and poor prognosis [3][4][5][6]. VAP-prevention strategies have recently been grouped together into care bundles. These strategies, including hand hygiene, daily sedation interruption, protocolized weaning evaluation, and semirecumbent positioning, have been shown to effectively reduce rates of VAP in ICU in Taiwan and other areas [7][8][9]. Although a few novel antibiotics against CR-GNB were developed in recent years, their activity varied in GNB with differing resistance mechanisms [10]. Moreover, some novel agents are not available in many countries. Combination therapy is generally recommended for CR-GNBrelated infection with moderate-to-high severity [11][12][13]. However, the optimal combination regimen remains unknown.
Colistin is effective against many GNBs and poses a high sensitivity rate against CR-Acinetobacter baumannii complex (CRAB), CR-enterobacteriaceae (CRE), and CR-Pseudomonas aeroginosa (CRPA). Colistin exerts its bactericidal effects by disrupting the integrity of the outer membrane, resulting in leakage of the cytoplasmic content, and neutralization of GNB endotoxins [14]. In the past, a before-after prospective study suggested that the daily intratracheal administration of colistimethate sodium at a dose of 1.6 million international units during 2 weeks, could significantly reduce the incidence of Gram-negative nosocomial bronchopneumonia in a population of critically ill patients on mechanical ventilation [15]. Despite its increasing use for managing nosocomial infection, administering intravenous (IV) colistin is frequently limited by its adverse reactions, especially nephrotoxicity [16]. Meanwhile, the low penetration of colistin in lung tissue may lead to inadequate antibacterial activity of intravenous colistin [17,18].
Nebulization has been shown to increase colistin concentration in lung tissue effectively [17]. The role of adjunctive nebulized colistin remains controversial in most recent HAP/VAP guidelines and CR-GNB treatment guidance due to limited evidences from randomized clinical trials [4,19]. Nebulized colistin in substitutive strategy, which means nebulized colistin without intravenous colistin [20], has also been used occasionally, especially in patients with impaired renal function and concerns of nephrotoxicity related to intravenous colistin. However, clinical evidences regarding the effectiveness of substitutive nebulized colistin are more scarce. This retrospective study included critically ill patients with nosocomial pneumonia caused by CR-GNB. We aimed to investigate the impact of substitutive nebulized colistin in the treatment outcomes of these patients. The differences in treatment outcomes between high-and low-dose substitutive nebulized colistin was analyzed as well.

Patients and settings
This is a retrospective cohort study conducted in a referral medical center in Taiwan. From January 2016 to December 2019, ICU-admitted patients with nosocomial pneumonia caused by colistin-susceptible CR-GNB and did not receive intravenous colistin were eligible for enrollment. The inclusion criteria included: (a) ICUadmitted patients diagnosed with nosocomial pneumonia developing more than 48 h after admission. (b) Positive cultures of CR-GNB isolated from respiratory specimens, including sputum, tracheal aspirates, and bronchoalveolar lavage fluid. Exclusion criteria included: age < 20 years, diagnosis of community acquired pneumonia or healthcare associated pneumonia, concomitant lung cancer with obstructive pneumonitis, CR-GNB that were resistant to colistin, did not receive anti-CRGNB antibiotics (including sulbactam, tigecycline, and carbapenem), and received intravenous colistin. The study protocol was approved by the Institutional Review Board of Taipei Veterans General Hospital and informed consent form was waived. (2020-11-006AC).

Diagnosis of pneumonia and microbiological tests
The diagnosis of pneumonia was based on new or progressive infiltrates in the chest radiograph, in addition to at least two clinical findings suggestive of pneumonia. The suggestive clinical findings included exacerbated cough, increased production of purulent sputum, fever (≥ 38 °C) or hypothermia (< 35 °C), and leukocytosis (white cell count ≥ 10,000/cumm) or leukopenia (white cell count < 4000/cumm). HAP was defined as pneumonia occurring ≥ 48 h after hospital admission. Whereas, VAP was defined as pneumonia developing ≥ 48 h after endotracheal intubation with invasive mechanical ventilator. Causative organisms were defined as CR-GNB isolated from respiratory specimens, including sputum, endotracheal aspirates (moderate or heavy growth by semiquantitative method), and bronchoalveolar lavage fluid with a concentration of ≥ 10 4 colony-forming units/ mL. The collection date of the index culture study was defined as the pneumonia index date.

Data collection and definitions
Demographic characteristics and underlying comorbidities were obtained from hospital chart reviews. The disease severities were evaluated using the Acute Physiology and Chronic Health Evaluation (APACHE) II score on the day of ICU admission, Sequential Organ Failure Assessment (SOFA) scores on the pneumonia index date, and the presence of organ dysfunction upon pneumonia diagnosis, including septic shock (vasopressor use), renal failure (under dialysis), and respiratory failure (with mechanical ventilator and PaO2/FiO2 ratio < 200).

Microbiological tests and treatment regimens
The results of susceptibility tests to carbapenems from the cultured isolates were determined based on the Clinical and Laboratory Standards Institute recommendations [21]. The isolates were considered susceptible to colistin if the minimum inhibitory concentrations were ≤ 2 mg/L in the broth microdilution method.
All the patients were not treated with intravenous colistin. Nebulized colistin administered during the treatment course of nosocomial pneumonia with a duration ≥ 2 days were recorded. Substitutive nebulized colistin was defined as nebulized colistin in conjunction with non-colistin intravenous antibiotics. The daily dose of substitutive nebulized colistin ranged from 2 million IU (MIU) colistimethate sodium (CMS) (66.8 mg colistin) to 15 MIU CMS (501 mg colistin) given 2 or 3 times a day. For dosage strategy analysis, we defined low-dose nebulized colistin as a daily dose ≤ 6 MIU CMS (200.4 mg colistin), and high-dose nebulized colistin as > 6 MIU CMS. Details of nebulized colistin preparation are shown in materials and methods of Additional file 1.
Intravenous antibiotics administered within 7 days of the pneumonia index date with ≥ 2-day duration were recorded. Inappropriate empirical therapy was considered when the patient did not receive at least one in vitro active antimicrobial agent within 24 h of pneumonia onset [22]. Meropenem was considered an active agent if it was prescribed in a dosage of 6 g/day in isolates with MIC ≤ 8 mg/L. VAP prevention bundle, including hand hygiene, oral hygiene, daily sedation interruption, and head-of-bed elevation, is widely adopted in all the ICUs in the study hospital.

Outcome evaluation
The primary endpoint was clinical responses on days 7, 14, and 28. Clinical responses were classified as cure (resolution of symptoms/signs of pneumonia, improvement or lack of progression of chest radiographic abnormalities, and antibiotics-free), improvement (substantial improvement of symptoms/signs of pneumonia, improvement or lack of progression of chest radiographic abnormalities, but remaining on antibiotic therapy), and failure (no apparent response to therapy, persistent or worsening of symptoms/signs of pneumonia, persistent or progression of radiographic abnormalities that required additional antibiotic therapy, or death) [23]. The secondary endpoints included all-cause mortality at day-14 and day-28, ventilator weaning rate at day-28, and new-onset dialysis at day-28. Details of clinical outcomes evaluation are provided in materials and methods of Additional file 1.

Time window bias adjustment and propensity score matching
We created a second cohort for the adjustment of timewindow bias related to delayed initiation of nebulized colistin [24]. Patients who died within 3 days of the onset of pneumonia, or who started nebulized colistin more than 3 days of the pneumonia index date, were excluded. After time-window bias adjustment, a propensity score (PS)-matched cohort was built using a PS approach with 1:1 matching and caliper width of 0.2 applied to both patients with and without nebulized colistin [25]. PS were created using logistic regression as a function of age, sex, smoking, pathogens, pneumonia types, ICU types, comorbidities, APACHE II scores, SOFA scores, albumin levels, organ failure, and key intravenous antibiotic used against CR-GNB. A PS-matched cohort was also made between patients with high-and low-dose nebulized colistin for sub-group analysis.

Statistical analysis
Statistical analyses were performed using the SPSS version 25.0 software (SPSS, Inc., Chicago, IL, USA). Continuous variables were compared by the Mann-Whitney U test, and categorical variables with Pearson's chisquare or Fisher's exact tests, as appropriate. We used mean imputation for missing data.
Regarding treatment outcomes, clinical failure rate, allcause mortality, ventilator weaning, and new-onset dialysis, were compared between patients with and without substitutive nebulized colistin; and patients with highand low-dose nebulized colistin, both in original cohort and PS-matched cohort with time-window bias adjustment. Binary logistic regression analysis with stepwise selection was performed to determine the independent variables associated with clinical failure. All variables with p value < 0.1 in the univariate analysis were included in the multivariate model. All tests were two-tailed and a p value < 0.05 was considered statistically significant.
Compared to patients without nebulized colistin, those with nebulized colistin were older, more likely to smoke, and be infected by CRAB. They also had medical ICU admission, renal insufficiency, diabetes, autoimmune diseases, lower APACHE II score upon ICU admission, dialysis after developing pneumonia, higher serum albumin level, lower CRP level, and were less likely to have a PF ratio < 200. Patients with nebulized colistin were more likely to receive intravenous sulbactam and tigecycline, but were less likely to be treated with carbapenem. The median duration of nebulized colistin treatment was 7 days (IQR: 6-14 days) and the median daily dose was 6 MIU CMS (IQR 4-15 MIU).

PS-matched cohort after time-window bias adjustment
After time-window bias adjustment and PS matching, we built a PS-matched cohort that included 115 patient pairs. As shown in Table 2, there were no significant differences in demographics, underlying comorbidities, disease severity, antibiotics used, or laboratory results   between patients stratified according to nebulized colistin treatment in the PS-matched cohort.

Substitutive nebulized colistin was associated with a better treatment outcome
As shown in Table 3   colistin had a significantly lower 28-day mortality risk compared to patients without substitutive nebulized colistin (log rank p = 0.005). The curves separated early after pneumonia onset. In the PS-matched cohort, there was no significant difference in 28-day mortality risk between patients with and without substitutive nebulized colistin. Microbiological eradication rates on day 14, and day 28 were significantly higher in patients with nebulized colistin in both the original and PS-matched cohorts. (Additional file 1: Table S1).

Independent factors associated with treatment outcomes
Univariate and multivariate analyses were performed to identify independent clinical factors associated with clinical failure at day 14. As shown in

Impact of dosage of substitutive nebulized colistin on treatment outcomes
We performed sub-group analysis to investigate the impact of nebulized colistin dosage in treatment outcomes. The demographics of patients with high-(> 6 MIU/day) and low-dose (≤ 6 MIU/day) nebulized colistin in the original and PS-matched cohorts is shown in Additional file 1: Table S2. In the original cohort, as compared to patients with high dose nebulized colistin, those with low doses were more likely to be male, have HAP, have diabetes, and were less likely to be treated with carbapenem and tigecycline. The median daily dosage of nebulized colistin were 4 MIU and 15 MIU, and median treatment duration were 7 and 8 days, in patients with low and high dose nebulized colistin, respectively. In the PS-matched cohort with 124 patient pairs, there were no significant differences in demographics between patients with low-and high-dose nebulized colistin. Comparisons of treatment outcomes between patients with low-and high-dose nebulized colistin are shown in Additional file 1: Table S3. In the original cohort, patients with high-dose nebulized colistin had higher clinical failure rate on days 7, 14, and 28, as well as higher 28 days and hospital mortalities. In the PS-matched cohort, there were no differences in clinical failure and mortality rates between patients with low-and high-dose nebulized colistin. The 28-day ventilator weaning and occurrence rates of 28-day new-onset dialysis were also comparable between the two groups.
Kaplan-Meier analyses of all-cause mortalities between patients with high and low dose nebulized colistin in the original and PS-matched cohorts are shown in Additional file 1: Figure S1. In the original cohort, patients with low dose nebulized colistin had significantly lower 28-day mortality risk compared to patients with high dose nebulized colistin (log rank p = 0.004). In the PS-matched cohort, there was no significant difference in Sub-group analysis was performed to identify specific populations that may benefit from high-dose nebulized colistin in the PS-matched cohort. As shown in Fig. 3, no significant differences in clinical failure rate were found in all the sub-group of patients. However, patients with age ≥ 75 years, smoker, with chronic lung disease, with APACHE II score ≥ 20, and with tigecycline treatment, may benefit more from high-dose nebulized colistin with a trend of lower clinical failure rate.

Discussion
This retrospective cohort study included critically ill patients with HAP/VAP caused by CR-GNB and investigated the clinical efficacy of substitutive nebulized colistin. In the multivariate analysis in the PS-matched cohort, we found that substitutive nebulized colistin is an independent factor associated with fewer cases of day 14 clinical failure. Meanwhile, substitutive nebulized colistin was not associated with new-onset dialysis. We also found that patients treated with high-and low-dose substitutive nebulized colistin had similar treatment outcomes, including clinical failure and all-cause mortality rates.
Colistin is widely used as one of the key agents against CR-GNB. Adverse reactions, especially nephrotoxicity, and low concentrations in lung tissue seriously limited its use for critically ill patients with HAP or VAP [16,17,26]. Colistin is a concentration-dependent antibiotic with a post-antibiotic effect, which makes it particularly suitable for nebulization as high lung concentrations can be expected [27]. Adjunctive nebulized colistin has been shown to improve clinical and microbiological responses in patients with pneumonia caused by CR-GNB [28][29][30][31]. However, due to insufficient randomized controlled trials, the role of adjunctive nebulized colistin in pneumonia caused by CR-GNB remains controversial in the latest treatment guidelines [4,[11][12][13]19].
Substitutive nebulized colistin refers to nebulized colistin without concomitant intravenous colistin. It is sometimes prescribed in patients with renal insufficiency or when nephrotoxicity is a concern, especially in ICUadmitted patients. Studies related to substitutive nebulized colistin is very limited. A randomized, single-blind study compared the efficacy of substitutive nebulized colistin with intravenous colistin in patients with community acquired pneumonia caused by multidrug-resistant GNB [32]. They reported that substitutive nebulized colistin had comparable treatment effects with intravenous colistin and had a lower risk of nephrotoxicity. Kuo et al. conducted a small retrospective study on patients with pneumonia caused by multidrug-resistant Acinetobacter baumannii (MDRAB) [33]. They found that substitutive nebulized colistin was significantly associated with MDRAB eradication as compared with matched control that received non-colistin antibiotics [33]. In this retrospective cohort study, we included more than 500 patients with HAP/VAP caused by CR-GNB, and created a PS-matched cohort that included 115 patient pairs. We found that patients given substitutive nebulized colistin were associated with less clinical failure as compared to those without substitutive nebulized colistin, although the mortality rate were comparable between the two groups of patients. We also reported similar incidence rates of new onset dialysis, which, again, demonstrated the low risk of nephrotoxicity in nebulized colistin.
The optimal daily dose and treatment duration for nebulized colistin remains unclear. The mean daily dose of nebulized colistin ranged from 1.25 to 15 MIU and the mean duration of treatment ranged from 7 to 17.5 days in previous studies [32][33][34][35][36]. Due to concerns for heterogeneously distributed inhaled antibiotics in lung tissues and possible low drug deposition in the affected lobe [37], we speculated that patients with pneumonia may benefit more from higher doses of nebulized antibiotics. However, there were no direct comparisons regarding treatment outcomes between different dosing strategies of nebulized colistin to date. To the best of our knowledge, this is the first study evaluating the impact of nebulized colistin dosage in treatment outcomes of pneumonia. Possibly due to more comorbidities and more organ dysfunction in high dose group, in the original cohort, patients treated with high daily doses nebulized colistin had worse treatment outcomes. After PS matching, patients treated with high and low daily doses had comparable treatment outcomes. Interestingly, we found that patients with older age, smoking habit, chronic lung diseases, lower PF ratio, and concomitant tigecycline treatment, may have less clinical failure when using high dose substitutive nebulized colistin. Although the evidences remain weak, our findings suggested that the dosing strategy of nebulized colistin may affect treatment outcomes in specific populations of patients with nosocomial pneumonia.
This study had several limitations. First, as a retrospective cohort study, there are marked differences in the demographic characteristics and disease severities. The survival time bias related to late initiation of nebulized colistin may overestimate its benefit in mortality rate. However, we have established a PS-matched cohort after adjusting survival time bias to minimize confounders in our analysis. Second, there is no standardized protocol for nebulized colistin, including dosage and duration, in our enrolled patients [38]. However, it provided us the opportunities to elucidate the dosage issues of nebulized colistin dosage on treatment outcomes of these critically ill patients. Third, nebulized colistin was performed with jet nebulizer, not mesh nebulizer. Jet nebulizer may lead to lower lung deposition of colistin and underestimate the clinical efficacy of nebulized colistin [39]. However, all the enrolled cases had jet nebulizers in the standard procedure in this single center study. Finally, this study was conducted in a tertiary medical center specialized in critical care, and only HAP/VAP caused by CR-GNB were included for analysis. Therefore, caution should be exercised in applying our present findings in other clinical settings with non-CR-GNB pathogens.

Conclusions
This retrospective cohort study included critically ill patients with HAP/VAP caused by CR-GNB. A majority of the patients were infected by CRAB. As compared to patients treated with non-colistin regimen, patients treated with substitutive nebulized colistin had lower clinical failure rate and similar mortality rate. We also found that patients treated with high-and low-dose nebulized colistin had comparable treatment outcomes and nephrotoxicity risk. Sub-group analysis suggested that patients with older age, with smoking habits, with chronic lung disorders, and with higher severities may benefit more from high-dose nebulized colistin. Our findings are only applicable in settings with endemic HAP/ VAP caused by CR-GNB, mainly CRAB. Further prospective study is deserved to elucidate the clinical benefits of substitutive nebulized colistin, including the application of high dose nebulized colistin in specific population.